PROJECT ABSTRACT Stroke, the fifth leading cause of death and leading cause of long-term disability in the United States, has limited therapeutic options. Even with the advent of reperfusion therapies including tissue plasminogen activator (tPA) and mechanical thrombectomy, extensive injury from stroke often results from ischemia- reperfusion (IR), which damages the blood-brain barrier (BBB), the vessel network separating the brain from the circulatory system. IR causes biphasic openings in the BBB, the first occurring within several hours of insult and the second at 24-74 hours after stroke. The latter is generally irreversible and, thus, the most damaging. Clinically, stem cell therapy offers great promise for treating stroke, but is currently aiming for stroke rehabilitation by delivering cells during the recovery (not subacute) phase. Here, we propose a novel approach to administer neural stem cells (NSCs) in the sub-acute phase to limit early-stage BBB injuries, an outcome that would protect against the second phase of stroke damage. We base this proposal on our extensive and novel preliminary and pilot data derived from a stroke mouse model showing that human(h)NSCs transplanted into the brain 24h post-IR improves neurological function and reduces BBB damage. Further, we have demonstrated that a protein fragment of the brain extracellular matrix (ECM) component perlecan, termed domain V (DV), is neuroprotective after experimental ischemic stroke, and may represent a promising new stroke therapy. Intriguingly, preliminary results also suggest that DV enhances NSC survival and differentiation in to neurons in vitro. Therefore, in this study, we will test the hypothesis that NSCs, in combination with the neuroprotective and neuroreparative protein perlecan DV, will synergistically ameliorate pathophysiology and neurological outcome in stroked mice. Ameliorating BBB damage before NSC transplantation using a neuroprotectant DV will improve the brain environment for NSC survival and allow for greater NSC efficacy. We will employ a filament MCAO/reperfusion (IR injury) mouse model that mimics ischemic stroke injuries seen in patients. Since aging is a strong risk factor for stroke, we will use both young adult and aged female and male mice, in whom neurobehavioral deficits are found to be worse. Aim 1 will determine the effects of neural stem cells and DV co-administration on sub-acute stroke injury in young adult and aged mice. Aim 2 will determine the effects of sub-acute neural stem cell delivery and DV co-administration on neuro-repair and long-term stroke recovery. Aim 3 will investigate the direct effect of perlecan DV in mechanisms of a2b1-induced NSC neuronal differentiation in vitro. This study is significant because it will generate new preclinical data that demonstrate the optimal strategy for NSC treatment, coupled with a novel neuroprotectant for ischemic stroke. The study will use innovative methods by employing and combining adjuvant pharmacological treatment (neuroprotectant) with NSCs, to improve stroke outcome.